A critical aspect of tumor physiology is the sensation of oxygen in the microenvironment. Several studies have demonstrated that over-expression of the oxygen-responsive Hypoxia-Inducible Factor (HIF-1a ) protein in breast cancer correlates with poor prognosis, increased risk of metastasis and decreased survival. Moreover, hypoxic regions of tumors are a prominent source of tumor cells that are resistant to radiation and chemotherapy. It has also been proposed that hypoxia stimulates expansion of normal and cancer stem cells (CSCs). Yet, the specific contribution of HIF-1a to breast cancer progression and metastasis in relationship to CSCs remains undefined. Our long-term goal is to elucidate how hypoxia, through HIF-1a , regulates breast CSC/progenitor populations. The specific hypothesis that will be tested is that HIF-1a promotes CSC renewal, expansion and tumor-initiating ability through interactions with the Wnt/-catenin signaling pathway, which potentiates the HIF-1a transcriptional response, resulting in increased primary tumor growth and metastatic potential. This will be tested in three specific aims. 1) To determine if HIF-1a promotes breast CSC renewal and tumor-initiating activity and if hypoxic exposure enhances CSC activity. Using novel HIF-1a wild type (WT) and null (KO) tumor cells derived from the MMTV-PyMT mouse model of breast cancer, we will address if hypoxia acts through HIF-1a to promote breast CSC renewal by comparing tumorsphere formation efficiencies and the frequency of tumor initiating cells in WT or KO cells that are sorted for cell surface markers that characterize murine mammary CSCs. 2) To determine if HIF-1a interacts with the Wnt/-catenin signaling pathway to regulate breast CSC behavior. We will determine if -catenin activity is necessary for CSC activity in PyMT tumor cells, if HIF-1a and -catenin physically interact and if Wnt/-catenin regulation of CSC activity is HIF-1a dependent by using TS and limiting dilution transplantation assays. 3) To investigate if HIF-1a -regulated CSC populations are enriched for metastasis and to investigate how HIF-1a promotes colonization and metastasis. Flow-sorted sub-populations of CSCs found to be regulated by HIF-1a in Aim 1 will be tested for enriched metastatic potential compared to parental cells using an intracardiac injection model of experimental metastases. Lentiviral transduction will be used to determine the contribution of HIF-1a regulated target genes to these processes. If our hypotheses that HIF-1a promotes breast tumor growth and metastasis through enhancing CSC activity via interactions with the Wnt/-catenin are confirmed, then this could have significant impact on treatment paradigms, and would suggest that it may be possible to further refine treatments to specifically eradicate the breast CSC population through a combination of anti-HIF and anti- -catenin therapies. In addition, since the tumor-initiating niche may exist in a hypoxic microenvironment, it may also be possible to utilize anti-HIF therapies in combination with other adjuvant therapies to prevent metastatic spread or, in the future, for chemoprevention.